The research interests of my laboratory are centered on the molecular details of animal RNA virus replication and the interactions of these viruses with their vertebrate and invertebrate hosts. The experiments detailed in this proposal concern our current studies on Sindbis virus (SIN), type virus of the family Togaviridae. Several members of this family are agents of chronic arthropod-transmitted world health problems, and our long range goals include a thorough understanding of viral RNA replication and gene expression, virion assembly, and viral pathogenesis and immunity. We eventually hope to apply this information for design for antiviral therapies and effective vaccines. our previous in vivo studies on the assembly of functional RNA replication complexes have led to the development of an in vitro RNA replication system which is dependent upon addition of exogenous SIN plus-strand RNA. This system will be used to define RNA promoter elements and RNA-protein interactions critical for initiation of minus-strand synthesis, to test a current model for the regulation of SIN RNA replication and transcription, and to provide an assay for purification and characterization of the viral RNA-dependent RNA polymerase. A second aim is to elucidate and study the viral functions responsible for vertebrate host cell death. Preliminary work has demonstrated that stable mammalian cell lines can be isolated after transformation with SIN replicons expressing a dominant selectable marker. Genetic analysis of these adapted replicons should provide clues as to the viral elements responsible for shut off of host translation and transcription and induction of apoptosis. Relevant viral components will be used to identify interacting host components in an attempt to obtain a mechanistic understanding of how these processes occur. Incorporation of such adaptive mutations into SIN expression vectors should greatly extend their utility by minimizing perturbation of host-cell biology. A third major focus is to study the mechanisms by which two novel SIN isolates invade the CNS of weanling mice. Infectious clones have been constructed for closely related noninvasive/invasive pairs and the genetic loci responsible for neuroinvasion have been determined. For one neuroinvasive strain, peripheral infection appears to induce the production of a soluble mediator of blood-brain barrier breakdown. Elucidation of the mechanisms leading to mediator production as well as its characterization, possible purification,a nd identification could have potential applications for drug delivery to the CNS or, if this is a general mechanism used by other pathogens for CNS invasion, methods for neutralization of the mediator may be useful for preventing spread to the CNS.